Bacterial extracts often show anti-tumor effect. It was only recently discovered that the active component in the ill-defined cancer treatment is bacterial DNA, not protein, carbohydrate, or lipid. In contrast to human genomic DNA, bacterial DNA shows unmethylated cytidine-guanidine dinucleotide sequences, commonly referred to as CpG. Our own body's host defense system has evolved such a way that whenever CpG is found in the body, it serves as a "danger" signal. Resulting innate immunity can be harnessed to fight against tumor growth. Most attractively, once tumor-associated antigens are shed from the tumor, dendritic cells activated by CpG can process them and present to T lymphocytes that can kill the tumor cells. In short, CpG-based immunotherapy can exploit not only its innate immune response but also subsequent adaptive immune response. Thus CpG has become one of the most exciting immunotherapies in recent years. However, the approach works only with intra-tumoral or peri-tumoral route of administration in animal models. In Phase II human studies, intravenous route was ineffective while subcutaneous administration produced only minor effects. Taken together these rather disappointing outcomes can be attributed to poor delivery of the CpG in vivo. Recently we were able to demonstrate a way to circumvent this critical problem in delivery. Our approach was based on the fact that immune complexes can circulate in the blood for a long time so long as they do not trigger antigen crosslinking. In tumor-bearing mice our formulation showed a half-life of several days in the blood and as much as 20% of the dose accumulated in tumor tissue. In accordance with this favorable pharmacokinetic, intravenously administered CpG successfully retarded solid tumor growth. More recently we also discovered that a subcutaneous route was as effective. We have our own explanations of these exciting results and this proposal is designed to test these hypotheses. This portion constitutes one aspect of the proposal. The other portion of the proposal is concerned with development of two new systems that will not require a step that may be acceptable in animal studies but may not be in humans. Successful outcome of the proposed studies will undoubtedly accelerate CpG-based immunocancer therapy and will provide another viable option in treating cancer along with other existing treatments. Public Health Relevance: It has been known for many years that bacterial DNA fragments exert anti-tumor effect. To be effective, they must be introduced where tumor is growing. This proposal deals with strategies for developing a convenient intravenous or subcutaneous route of administration that will deliver the agents to the tumor tissue.